CN106167521A - A kind of solid phase synthesis process of Exenatide - Google Patents
A kind of solid phase synthesis process of Exenatide Download PDFInfo
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- CN106167521A CN106167521A CN201610739539.8A CN201610739539A CN106167521A CN 106167521 A CN106167521 A CN 106167521A CN 201610739539 A CN201610739539 A CN 201610739539A CN 106167521 A CN106167521 A CN 106167521A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
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Abstract
The present invention relates to the new synthetic method of a kind of Exenatide; with MBHA/AM resin as initiation material; synthesize with linker, aminoacid or dipeptides, tripeptides for primary raw material; carry out condensation with DIC/HoBt, DIC/HoAt for condensation reagent and extend peptide chain; use deprotecting regent removing Fmoc group; repeat amino acid couplings; de-Fmoc; amino acid couplings; the process of de-Fmoc obtains the peptide resin of Exenatide; the Exenatide peptide resin of cracking full guard obtains exenatide crude product, and last high performance liquid chromatography is purified the finished product obtaining Exenatide.The present invention passes through Fmoc (Hmb) Leu21The introducing of OH effectively alleviates peptide chain Ser11‑Val19Between the situation of β-pleated sheet, and by Fmoc Pro36‑Pro37‑Pro38The introducing of OH fragment effectively reduces and progressively connects Pro36‑Pro37‑Pro38During the impurity of De Pro, finally use the introducing of Fmoc His Gly OH to decrease (D His1) and (De Gly2), the impurity of (+Gly), thus difficulty when reducing isolated and purified, substantially increase product yield and purity.
Description
Technical field
The invention provides a kind of can the synthetic method of large-scale production Exenatide, particularly contribute to reducing racemization
With disappearance peptide and the formation of alleviation β-pleated sheet, belong to peptide synthesis technology field.
Background technology
Diabetes are a kind of being characterized with hyperglycemia caused by defect of insulin secretion or insulin action obstacle
Metabolic disease.Persistent high blood sugar and long-term metabolic are disorderly etc. may result in body tissue's organ, particularly eye, kidney, cardiovascular and
Neural infringement and dysfunction thereof and exhaustion.Severe patient can cause dehydration, electrolyte disturbance and acid base imbalance etc.
Acute complications ketoacidosis and Hyperosmotic coma.Whole world diabetics is more than 1.5 hundred million, and wherein type ii diabetes people exceedes
Half.
Exenatide, indication is that this product is for improving the glycemic control of type 2 diabetes mellitus patient, it is adaptable to alone diformazan is double
Guanidine, sulfonylurea. and metformin share sulfonylurea, blood glucose still controls the best patient.
The sequence of Exenatide is as follows:
His1-Gly2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Leu10-Ser11-Lys12-Gln13-Met14-
Glu15-Glu16-Glu17-Ala18-Val19-Arg20-Leu21-Phe22-Ile23-Glu24-Trp25-Leu26-Lys27-Asn28-
Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2
The at present synthesis of Exenatide mainly has three kinds of methods:
A: utilize gene recombination technology to obtain the sequence of Exenatide, disclose in patent CN1635117A, CN1693459A
This kind of method prepares the technology of Exenatide；
B: use the method synthesis of liquid phase synthesis or solid-liquid combination, but owing to the technics comparing of liquid phase synthesis is loaded down with trivial details, post processing fiber crops
Tired, the critical process point related to is too many, is therefore unfavorable for the synthesis of the polypeptide of gene comparision length.This method is in patent CN1018
35794A has open mentioning；
C: the method using solid phase synthesis, owing to each step of liquid phase synthesis will carry out separating-purifying, for 39 residues
For polypeptide, synthesis cycle is long, and the key parameter of middle process is too many, wayward, it is easy to cause the instability of technique,
Therefore the method synthesis Exenatide of solid phase synthesis is the most still used.Such as CN101538324, CN101357 in domestic patent
938.But owing to, during progressively coupling amino acid, owing to each amino acid whose characteristic is different, it exists in various degree
Side reaction, the aminoacid of especially conventional Fmoc protection, in connection procedure, there are the character of related substance and the character ten of target peptide
Tap is near, causes final purifies and separates difficulty to affect yield and product quality.
Summary of the invention
The present invention is directed to the deficiency in being conventionally synthesized, use aminoacid or dipeptides, the tripeptides small fragment of special construction, reduce
The difficult generation probability separating impurity in building-up process, can produce in order to large-scale.
Term illustrates:
Linker: the linking arm inserted with interlaminar resin at polypeptide, plays connecting peptides and improves the effect of resin properties.
Coupling reagent: aminoacid carboxyl and amino condensation can be caused to form the reagent of amido link.
Beta sheet: the secondary structure of polypeptide, plane of peptide band is folded into zigzag, between N-H and C=O of adjacent peptide chains main chain
Forming well-regulated hydrogen bond, in beta sheet, all of peptide bond is involved in the major axis of the formation of interchain hydrogen bond, hydrogen bond and beta sheet
Perpendicular relation.
Lysate: after Peptide systhesis completes, the examination that the cut-out of peptide side chain blocking group and peptide chain are discharged from resin
Agent.
Technical scheme is as follows:
The synthesis step of Exenatide:
1) with MBHA/AM resin as initiation material, synthesize with linker, aminoacid or dipeptides, tripeptides for primary raw material.
2) carry out condensation with DIC/HoBt, DIC/HoAt for condensation reagent and extend peptide chain, 20% piperidines/DMF solution or 0.1M
HoBt 20% piperidines/2%DBU/DMF solution be deprotecting regent remove Fmoc group.
Repeat amino acid couplings, de-Fmoc, amino acid couplings, the process of de-Fmoc obtains the peptide resin of Exenatide:
His-Gly-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Leu10-Ser
(tBu)-Lys(Boc)-Gln(Trt)-Met-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Val-Arg(pbf)-
(Hmb)Leu-Phe-Ile-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser
(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-rink amide AM resin/MBHA resin
Described each Fmoc-aminoacid, dipeptides, tripeptides consumption with the mol ratio of resin demand be: 2 ~ 6:1；Aminoacid and condensation
Reagent pre-activate pre-activate 5 ~ 15min under conditions of ice-water bath, reaction temperature controls at 20-35 DEG C, and the response time controls
30-150mins。
The peptide resin that above-mentioned steps obtains, after lysate cracks, obtains Exenatide through methyl tertiary butyl ether(MTBE) precipitation thick
Peptide.
Cutting liquid ratio is: TFA:ETD:H2O=(87.5 ~ 95): (10 ~ 2.5): (2.5 ~ 5)；Resin and the ratio of cutting liquid
Example is 6 ~ 15ml/g peptide resin, and lysate is pre-cooling 30mins at-10 ~-20 DEG C, and pyrolysis time is at 2.5 ~ 4hrs, cracking temperature
At 20 ~ 40 DEG C.
The preferred TFA:EDT:H of the present invention2O=90:5:5, lysate is pre-cooling 30mins at-20 DEG C, and pyrolysis time is
180mins, cracking temperature controls at 30 DEG C, and lysate and resin ratio are chosen as 8ml/g peptide resin, the lysate after filtration with
Methyl tertiary butyl ether(MTBE) ratio is 1:6(v/v).
The exenatide crude product obtained is obtained Exenatide finished product after preparative hplc purification lyophilizing.
Fmoc-His-Gly-OH.HCl is prepared as:
Being dissolved in anhydrous DCM by raw material Fmoc-His (Trt)-OH, add the EDCI of 1.5eq, HoSu under nitrogen protection, stirs
Mixing 22hrs, then wash, saturated common salt is washed, and organic facies anhydrous sodium sulfate is dried to obtain Fmoc-His (Trt)-Osu, will
Fmoc-His (the Trt)-Osu THF that obtains dissolves, and is added drop-wise to containing in the sodium bicarbonate aqueous solution of the H-Gly-OH of 1.2eq,
Reaction 3hrs, filters, and rotation is steamed.Then with phosphate aqueous solution regulation Ph to 3 ~ 4, DCM extraction, rear organic facies pickling, washing, saturated
Brine It, the hydrochloric ethyl acetate obtaining Fmoc-His (Trt)-Gly-OH 4N after drying processes 2.5hrs, is spin-dried for
After, cross chromatographic column and obtain Fmoc-His-Gly-OH.HCl.
The invention has the beneficial effects as follows: the method is with AM/MAHA resin as initial resin, then by Rink amide
Linker is as cross structure, then is sequentially connected with aminoacid or dipeptides, and the method for tripeptide fragment obtains the peptide resin of Exenatide, this
Invention effectively alleviates the situation of the β-pleated sheet between peptide chain Ser11-Val19 by the introducing of Fmoc-(Hmb) Leu21-OH, and leads to
The introducing crossing Fmoc-Pro36-Pro37-Pro38 OH fragment effectively reduces progressively connection Pro36-Pro37-Pro38 process
The impurity of middle De-Pro.The introducing finally using Fmoc-His-Gly-OH decrease (D-His1) and (De-Gly2), (+
Gly) impurity, thus difficulty when reducing isolated and purified, substantially increase product yield and purity.
Specific embodiment:
Below by way of example, the present invention is described, but, these examples are intended merely to the specific description present invention's in further detail
Concrete operations rather than limit the invention.
Embodiment 1
By raw material Fmoc-His (Trt)-OH(62g, 100mmol) be dissolved in anhydrous DCM (200ml), add EDCI (28.8g,
150mmol), HoSu (17.3g, 150mmol) under nitrogen protection, stirs 22hrs, then washing (60ml*3), saturated common salt
Washing (60ml*3), organic facies anhydrous sodium sulfate is dried to obtain Fmoc-His (Trt)-Osu (68.5g, yield: 95.6%).Will
Fmoc-His (the Trt)-Osu(95.6mmol obtained) with THF (100ml) dissolve, be added drop-wise to containing H-Gly-OH (9.75g,
In sodium bicarbonate (15.2g, 143.4mmol) aqueous solution (50ml) 114.7mmol), reacting 3hrs, filter, rotation is steamed.Then use
5% phosphate aqueous solution regulation pH value is to 3 ~ 4, and DCM extracts, and organic facies is washed, after drying through pickling, washing, saturated aqueous common salt subsequently
The hydrochloric ethyl acetate (100ml) of Fmoc-His (the Trt)-Gly-OH 4N obtained processes 2.5hrs, after being spin-dried for, through chromatography
Column purification, obtains Fmoc-His-Gly-OH.HCl (34.1g, 78.39mmol), yield 82%；
The preparation of the Exenatide peptide resin of example 2 full guard
(1) AM resin(9.4g, 5mmol, sub:0.53mmol/g are weighed) join in the glass reactor of jacketed, add
The swelling 2hrs of 100mlDCM, then sucking filtration, and wash 2 times with 2%DIEA/DCM solution, DMF washes twice, and adds Rink amide
Linker (5.4g, 10mmol), HoBt (1.35g, 10mmol), then with appropriate DMF dissolve after, add DIC (1.58ml,
10mmol), reacting 2.5hrs in the jacketed reactor of circulating water temperature 30 DEG C, reaction end 1,2,3-indantrione monohydrate detection liquid is examined
Survey, be negative, illustrate that reaction is completely.
After reaction completely, protect with deprotection loss of thick fluid, Gly-Gly-Pro-Ser (tBu)-Ser (tBu)-Gly-Ala-
Pro-Pro-Pro-Ser (tBu)-AM resin section 20% piperidines/DMF solution deprotection 30mins, His-Gly-Glu
(OtBu)-Gly- Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Leu10-Ser(tBu)-Lys(Boc)-
Gln(Trt)-Met-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Val-Arg(pbf)-(Hmb)Leu-Phe-Ile-
The HoBt 20% piperidines/2%DBU/ of Glu (OtBu)-Trp (Boc)-Leu-Lys (Boc)-Asn (Trt) section deprotection 0.1M
DMF solution deprotection 30mins.
Often walk amino acid whose coupling according to connected mode coupling in [0024], but coupling reagent and amino acid requirement exist every time
In ice-water bath about pre-activate 15min, then it is attached.
The aminoacid of coupling is successively:
Fmoc-Ser(tBu)-OH、Fmoc-Pro-Pro-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser
(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Gly-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-
Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-
Phe-OH、Fmoc-(Hmb)Leu-OH、Fmoc-Arg(pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu
(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、
Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-
OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-His-Gly-OH。
The full guard peptide resin preparing Exenatide is as follows:
His-Gly-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Leu10-Ser
(tBu)-Lys(Boc)-Gln(Trt)-Met-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Val-Arg(pbf)-
(Hmb)Leu-Phe-Ile-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser
(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-rink amide AM resin。
The preparation of the Exenatide peptide resin of example 3 full guard
Weigh AM resin(28.3g, 15mmol, sub:0.53mmol/g) join in the glass reactor of jacketed, add
The swelling 2hrs of 300mlDCM, then sucking filtration, and wash 2 times with 2%DIEA/DCM solution, DMF washes twice, and adds Rink amide
Linker (16.19g, 30mmol), HoBt (4.06g, 30mmol), then with appropriate DMF dissolve after, add DIC (4.75ml,
30mmol), reacting 2.5hrs in the jacketed reactor of circulating water temperature 30 DEG C, reaction end 1,2,3-indantrione monohydrate detection liquid is examined
Surveying, be negative, reaction is described completely, remaining amino acid whose connection is carried out even according to the coupling mode of Rink amide linker
Connect, but be that aminoacid is carried out about pre-activate 15min in ice-water bath condition every time, then carrying out coupling, to obtain Exenatide complete
Protection peptide resin:
His-Gly-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Leu10-Ser
(tBu)-Lys(Boc)-Gln(Trt)-Met-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Val-Arg(pbf)-
(Hmb)Leu-Phe-Ile-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser
(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-rink amide AM resin。
The preparation of the Exenatide peptide resin of example 4 full guard
Weigh MBHA resin(10g, 5mmol, sub:0.5mmol/g) join in the glass reactor of jacketed, add
The swelling 2hrs of 100mlDCM, then sucking filtration, and wash 2 times with 2%DIEA/DCM solution, DMF washes twice, and adds Rink amide
Linker (5.4g, 10mmol), HoBt (1.35g, 10mmol), then with appropriate DMF dissolve after, add DIC (1.58ml,
10mmol), reacting 2.5hrs in the jacketed reactor of circulating water temperature 30 DEG C, reaction end 1,2,3-indantrione monohydrate detection liquid is examined
Surveying, be negative, reaction is described completely, remaining amino acid whose connection is carried out even according to the coupling mode of Rink amide linker
Connect, but be that aminoacid is carried out about pre-activate 15min in ice-water bath condition every time, then carrying out coupling, to obtain Exenatide complete
Protection peptide resin:
His-Gly-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Leu10-Ser
(tBu)-Lys(Boc)-Gln(Trt)-Met-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Val-Arg(pbf)-
(Hmb)Leu-Phe-Ile-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser
(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-rink amide MBHA resin。
The preparation of the Exenatide peptide resin of example 5 full guard
Weigh MBHA resin(30g, 15mmol, sub:0.5mmol/g) join in the glass reactor of jacketed, add
The swelling 2hrs of 300mlDCM, then sucking filtration, and wash 2 times with 2%DIEA/DCM solution, DMF washes twice, and adds Rink amide
Linker (16.19g, 30mmol), HoBt (4.06g, 30mmol), then with appropriate DMF dissolve after, add DIC (4.75ml,
30mmol), reacting 2.5hrs in the jacketed reactor of circulating water temperature 30 DEG C, reaction end 1,2,3-indantrione monohydrate detection liquid is examined
Surveying, be negative, reaction is described completely, remaining amino acid whose connection is carried out even according to the coupling mode of Rink amide linker
Connect, but be that aminoacid is carried out about pre-activate 15min in ice-water bath condition every time, then carrying out coupling, to obtain Exenatide complete
Protection peptide resin:
His-Gly-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Leu10-Ser
(tBu)-Lys(Boc)-Gln(Trt)-Met-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Val-Arg(pbf)-
(Hmb)Leu-Phe-Ile-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser
(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-rink amide AM resin。
The preparation of the thick peptide of example 6 Exenatide
The preparation of lysate: configuration lysate 100ml, wherein trifluoroacetic acid 90ml, EDT (1，2-ethandithiol) 5ml, pure water
5ml, after shaking up ,-20 DEG C of cooling 30min are standby.
By the full guard Exenatide resin 10g (-20 DEG C of pre-cooling 30min) in example 2, join 250ml with magneton
Round-bottomed flask in, by [034] lysate take out shake up, measure 80ml and join in flask, cracking temperature is on 30min
It is raised to 30 DEG C, and maintains 30 ± 2 DEG C of stirring 2.5hrs.Filter with sand core funnel and use a small amount of cutting liquid washing resin.To merge
Filtrate slowly import cold methyl tertiary butyl ether(MTBE) (-20 DEG C of pre-cooling 2hr) 540ml, process of pouring into methyl tertbutyl to be kept is at the uniform velocity
, there is white precipitate in stirring, and with filtered on buchner funnel, and with cold methyl tertiary butyl ether(MTBE) washing filter cake 3 times, vacuum is drained and obtained slightly
Peptide 4.82g(yield: 92.1%)
The preparation of the thick peptide of example 7 Exenatide
The preparation of lysate: configuration lysate 100ml, wherein trifluoroacetic acid 90ml, EDT (1，2-ethandithiol) 5ml, pure water
5ml, after shaking up ,-20 DEG C of cooling 30min are standby.
By the full guard Exenatide resin 10g (-20 DEG C of pre-cooling 30min) in example 3, join 250ml with magneton
Round-bottomed flask in, by [037] lysate take out shake up, measure 80ml and join in flask, cracking temperature is on 30min
It is raised to 30 DEG C, and maintains 30 ± 2 DEG C of stirring 2.5hrs.Filter with sand core funnel and use a small amount of cutting liquid washing resin.To merge
Filtrate slowly import cold methyl tertiary butyl ether(MTBE) (-20 DEG C of pre-cooling 2hr) 540ml, process of pouring into methyl tertiary butyl ether(MTBE) to be kept is even
, there is white precipitate in speed stirring, with filtered on buchner funnel, and washs filter cake 3 times with cold methyl tertiary butyl ether(MTBE), and vacuum is drained and obtained
Thick peptide 4.75g(yield: 91.8%)
The preparation of the thick peptide of example 8 Exenatide
The preparation of lysate: configuration lysate 500ml, wherein trifluoroacetic acid 450ml, EDT (1，2-ethandithiol) 25ml, pure water
25ml, after shaking up ,-20 DEG C of cooling 30min are standby.
By the full guard Exenatide resin 50g (-20 DEG C of pre-cooling 30min) in example 4, join the 1L circle with magneton
In end flask, being taken out by the lysate in [040] and shake up, measure 400ml and join in flask, cracking temperature rises at 30min
To 30 DEG C, and maintain 30 ± 2 DEG C of stirring 2.5hrs.Filter with sand core funnel and use a small amount of cutting liquid washing resin.By merge
Filtrate slowly imports cold methyl tertiary butyl ether(MTBE) (-20 DEG C of pre-cooling 2hr) 2.7L, and process of pouring into methyl tertbutyl to be kept at the uniform velocity stirs
Mixing, white precipitate occur, with filtered on buchner funnel, and with cold methyl tertiary butyl ether(MTBE) washing filter cake 3 times, vacuum is drained and is obtained thick peptide
24.8g(yield: 93.3%)
The purification of example 9 exenatide crude product
The crude product weighing Exenatide dissolves with the aqueous acetic acid of 10%, and the solution microporous filter membrane filtration of 0.45 μm is treated
With.
The condition that high performance liquid chromatography is purified, chromatographic column is 10 μm with particle diameter, and aperture is the C18 bonded silica of 300
Glue is fixing phase.Change salt through 2 step purification one steps and obtain the finished product of Exenatide.Purity is 99.47%, [D-His1] impurity,
[De-Gly2] impurity, [+Gly2] impurity is respectively less than 0.1%.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a solid phase synthesis process for Exenatide, comprises the following steps that
1) Fmoc-His-Gly-OH.HCl is prepared: be dissolved in anhydrous DCM by raw material Fmoc-His (Trt)-OH, add 1.5eq
EDCI, HoSu under nitrogen protection, stir 10-24hrs；Then washing, saturated common salt is washed, organic facies anhydrous sodium sulfate
It is dried to obtain Fmoc-His (Trt)-Osu, Fmoc-His (the Trt)-Osu THF obtained is dissolved, is added drop-wise to containing 1.2eq
H-Gly-OH sodium bicarbonate aqueous solution in, react 2-6hrs, filter, rotation steam, then with phosphate aqueous solution regulation pH value to 3
~ 4, DCM extract, rear organic facies pickling, washing, and saturated aqueous common salt washs, and obtain Fmoc-His (Trt)-Gly-OH after drying and use
The hydrochloric ethyl acetate of 4N processes 2-4hrs, after being spin-dried for, crosses column chromatography and obtains Fmoc-His-Gly-OH.HCl；
2) the Exenatide peptide resin of full guard is prepared:
Selecting AM resin or MBHA resin is resin raw material, and selecting linker, aminoacid or dipeptides, tripeptides is main conjunction
Become raw material；It is that condensation reagent carries out condensation prolongation peptide chain by Fmoc-Linker with HBTU/DIEA, DIC/HoBt or DIC/HoAt
Or Rink amide linker is connected on AM resin or MBHA resin, use deprotecting regent removing Fmoc base
Group, repeats amino acid couplings, de-Fmoc, amino acid couplings, the process of de-Fmoc, progressively connects aminoacid or fragment, even successively
The aminoacid of connection is:
Fmoc-Ser(tBu)-OH、Fmoc-Pro-Pro-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser
(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Gly-Gly-OH、
Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu
(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-(Hmb)Leu-OH、Fmoc-Arg(pbf)-OH、Fmoc-Val-
OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-
Met-OH、Fmoc-Gln(Trt)-OH、
Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、
Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-His-Gly-OH；
With 1,2,3-indantrione monohydrate detection detection reaction end in connection procedure, 1,2,3-indantrione monohydrate detects into negative explanation and connects completely；Wherein, often walk
Amino acid whose coupling according to identical connected mode coupling, each coupling reagent and amino acid requirement pre-activate in ice-water bath,
It is attached again；
Finally obtain the full guard peptide resin of Exenatide: His1-Gly2-Glu3(OtBu)-Gly4-Thr5(tBu)-Phe6-
Thr7(tBu)-Ser8(tBu)-Asp9(OtBu)-Leu10-Ser11(tBu)-Lys12(Boc)-Gln13(Trt)-Met14-Glu15
(OtBu)-Glu16(OtBu)-Glu17(OtBu)-Ala18-Val19-Arg20(pbf)-(Hmb)Leu21-Phe22-Ile23-Glu24
(OtBu)-Trp25(Boc)-Leu26-Lys27(Boc)-Asn28(Trt)-Gly29-Gly30-Pro31-Ser32(tBu)-Ser33
(tBu)-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39(tBu)-rink amide AM resin/MBHA resin
3) crack the Exenatide peptide resin of full guard: by above-mentioned steps 2) in the full guard peptide resin warp of Exenatide that obtains
Cross lysate cracking, and with a small amount of cutting liquid washing resin, after after methyl tertiary butyl ether(MTBE) precipitation, obtain the thick of Exenatide
Product；
4) purifying Exenatide crude product: exenatide crude product is dissolved with the aqueous acetic acid of 10%, the solution micropore mistake of 0.45 μm
Membrane filtration, stand-by；Employing high performance liquid chromatography is purified, and changes salt through two-step purifying one step and obtains the one-tenth of Exenatide
Product.
The solid phase synthesis process of a kind of Exenatide the most according to claim 1, it is characterised in that: step 2) selected tree
The substitution value of fat is at 0.35-1.0mmol/g.
The solid phase synthesis process of a kind of Exenatide the most according to claim 1, it is characterised in that: step 2) connection procedure
Middle use coupling reagent is DIC/HoBt, DIC/HooBt or DIC/HoAt combination.
The solid phase synthesis process of a kind of Exenatide the most according to claim 1, it is characterised in that: step 2) connection procedure
In connect every time before by aminoacid pre-activate 5-15mins under ice bath, the response time is at 30mins ~ 150mins.
The solid phase synthesis process of a kind of Exenatide the most according to claim 1, it is characterised in that: step 2) connection procedure
Middle aminoacid, coupling reagent are (2:2:1)～(6:6:1) with the ratio of resin.
The solid phase synthesis process of a kind of Exenatide the most according to claim 1, it is characterised in that: step 2) in remove-insurance
Protect, wherein Gly-Gly-Pro-Ser (tBu)-Ser (tBu)-Gly-Ala-Pro-Pro-Pro-Ser (tBu)-NH2 section remove-insurance
Protect with 20% piperidines/DMF solution deprotection 10-30 min；His-Gly-Glu(OtBu)-Gly- Thr(tBu)-Phe-Thr
(tBu)-Ser(tBu)-Asp(OtBu)-Leu10-Ser(tBu)-Lys(Boc)-Gln(Trt)-Met-Glu(OtBu)-Glu
(OtBu)-Glu(OtBu)-Ala-Val-Arg(pbf)-(Hmb)Leu-Phe-Ile-Glu(OtBu)-Trp(Boc)-Leu-Lys
(Boc) HoBt 20% piperidines of-Asn (Trt) section deprotection 0.1M/2%DBU/DMF solution deprotection 20-30mins.
The solid phase synthesis process of a kind of Exenatide the most according to claim 1, it is characterised in that: lysate in step 3)
Proportioning be TFA:ETD:H2O=(87.5 ~ 95): (10 ~ 2.5): proportioning TFA:ETD:H2O=of (2.5 ~ 5), preferably lysate
90:5:5。
The solid phase synthesis process of a kind of Exenatide the most according to claim 1, it is characterised in that: cutting liquid in step 3)
It is 8ml/g peptide resin with the ratio that the ratio of resin is 6 ~ 15ml/g peptide resin, preferably cutting liquid and resin.
The solid phase synthesis process of a kind of Exenatide the most according to claim 1, it is characterised in that: lysate in step 3)
Pre-cooling 30mins at-10 ~-20 DEG C, pyrolysis time is at 2.5 ~ 4hrs, and cracking temperature is at 20 ~ 40 DEG C；Be preferably lysate-
Pre-cooling 30mins at 20 DEG C, pyrolysis time is at 180mins, and cracking temperature controls at 30 DEG C.
The solid phase synthesis process of a kind of Exenatide the most according to claim 1, it is characterised in that: in step 4) efficiently
The condition of liquid chromatography be chromatographic column be 10 μm with particle diameter, aperture be the C18 bonded silica gel of 300 be fixing phase.
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| CN105294853A (en) * | 2015-11-13 | 2016-02-03 | 中肽生化有限公司 | Preparation method for liraglutide |
| CN110372784A (en) * | 2019-07-29 | 2019-10-25 | 深圳佳肽生物科技有限公司 | The synthetic method and application of Exenatide |
| CN110964097A (en) * | 2018-09-28 | 2020-04-07 | 南京华威医药科技集团有限公司 | Synthesis of exenatide by solid-phase fragment method |
| CN111349153A (en) * | 2020-04-10 | 2020-06-30 | 四川吉晟生物医药有限公司 | Preparation method of atrial natriuretic peptide |
| CN112111001A (en) * | 2019-06-19 | 2020-12-22 | 翰宇药业（武汉）有限公司 | Method for synthesizing thymosin T alpha-1 |
| US11518794B2 (en) | 2016-08-19 | 2022-12-06 | Shenzhen JYMed Technology Co., Ltd. | Synthesis method for liraglutide with low racemate impurity |
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| CN111349153A (en) * | 2020-04-10 | 2020-06-30 | 四川吉晟生物医药有限公司 | Preparation method of atrial natriuretic peptide |
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